<i>In vivo</i> evolution of resistance to artesunate + mefloquine.

<p>Artesunate (ATN) and mefloquine (MF) were given together to <i>P. chabaudi</i>-infected mice over many generations. After twenty seven sub-inoculations under increasing ATN + MF exposure, the drug-resistant population was cloned. One of these clones, denoted AS-ATNMF1, was selected from subsequent studies.</p

Drug resistance lineage of <i>Plasmodium chabaudi</i> AS.

<p>Note: the Figure does not depict all drug-resistant clones within the AS lineage. Only those relevant to the present work are represented. Each drug used to select resistant clones is noted inside each arrow and the increasing colour tonality of the arrow represents an approximation of the increase in drug doses during the evolution of resistance. The length of each arrow depicts the approximate relative time for generating the resistance phenotype. The generation of pyrimethamine resistance was a result of a single-step selection, whilst for all other drugs the evolution of resistance resulted from prolonged exposures to small increments in drug concentrations over many generations. The genotypic differences between a particular parasite and its progenitor are noted in brackets. Asterisks depict the clones used in this work.</p

Oligonucleotide primers and TaqMan probes used to ascertain <i>mdr1</i> copy number and transcription levels.

<p><i>F</i>: forward; <i>R</i>: Reverse; <i>Tp</i>: TaqMan probe.</p

Drug test results.

<p>Each line represents the evolution of parasitaemia in each parasite clone from day 4 post-inoculum (p.i) in the absence of treatment (a), under artesunate + meloquine treatment (b), under mefloquine treatment (c) or under artesunate treatment (d). Each data point is a mean of % parasitaemias ± SE resulting from reads of groups of two and five mice in the untreated and treated groups, respectively.</p

Relationships between genomic DNA, transcript and protein amounts of <i>mdr1</i> between the different parasite clones.

<p>Values were determined by dividing the mean N-fold expression between each molecular species.</p

Typical Western blot result depicting MDR1 expression in the artesunate + mefloquine resistant clone AS-ATNMF1.

<p>Hybridization was carried out with α anti Pgh1 antibodies and α anti tubulin antibodies. AS-3CQ and AS-ATN were used as one-<i>mdr1</i> copy controls and AS-15MF was used as the two-<i>mdr1</i> copies control.</p

Differences in <i>mdr1</i> genomic DNA, cDNA and protein amounts between the different parasite clones.

<p>Columns represent mean of five independent experiments with corresponding Standard Error bars. Cases were differences in relative <i>mdr1</i> amounts between a particular parasite and its one copy comparator (AS-3CQ or AS-ATN) are statistically significant after Student's t-tests are represented by * (p≤0.01) or ** (0.05≥p≥0.01).</p

Innovation in healthcare manufacturing: transforming deployed medical care

This paper analyses the case for transforming deployed medical care through innovations in advanced manufacturing. Early-stage findings present expert views based on a cross-disciplinary roadmapping workshop, designed to steer requirements and identify desirable applications across a range of medical manufacturing engineering disciplines. This paper proposes how the adoption of Redistributed Manufacturing (RDM) has the potential to transform operational patient care pathways and supply chains in the context of military and emergency medicine. This encompasses the use of technologies such as additive manufacturing, bioprinting and other distributed approaches. This work is part of a national UK research programme and sets out the foundations for a position paper to guide future research and investment in both academia and practice

EBL Amino acid sequence alignment of various malaria species and <i>Plasmodium yoelii</i> strains, and predicted protein structure consequences of the C351Y polymorphism.

<p><b>(A)</b> EBL orthologous and paralogous sequences from a variety of malaria species and <i>P. yoelii</i> strains were aligned using ClustalW. Only the amino acids surrounding position 351 are shown. The cysteine in positon 351 in <i>P. yoelii</i> is highly conserved across strains and species, with only strain 17X1.1pp bearing a C to Y substitution. PchAS: <i>Plasmodium chabaudi</i> AS strain; PbANKA: <i>Plasmodium berghei</i> ANKA strain; Py17X/17X1.1pp/CU/YM: <i>P. yoelii</i> 17X,17X1.1pp,CU,YM strains; Pk-DBLα/β/γ: <i>Plasmodium knowlesi</i> Duffy Binding Ligand alpha/beta/gamma (H strain); PvDBP: <i>Plasmodium vivax</i> Duffy Binding Protein (Sal-I strain);PcynB_DBP1/2: <i>Plasmodium cynomolgi</i> Duffy Binding Proteins 1/2 (B strain); Pf3D7_EBA140/175/181: <i>Plasmodium falciparum</i> Erythrocyte Binding Antigens 140/175/181 (3D7 strain). <b>(B)</b> Energy minimized homology model of the wild type <i>P. yoelii</i> (Py17XWT) Erythrocyte Binding Ligand (EBL). Inset depicts the disulfide bond between C351 and C420. (The protein is represented in cyan and the disulfide bonds are in yellow). <b>(C)</b> Energy minimized homology model of the mutant (C351Y) <i>P. yoelii</i> (Py17X1.1pp) Erythrocyte Binding Ligand (EBL). Inset depicts the lack of a disulfide bond between Y351 (substituted C351) and C420. (The protein is represented in cyan and the disulfide bonds are in yellow and Tyr351 [mutated] is represented in magenta).</p

Genome-wide sequencing data.

<p><b>(A)</b> Genome-wide <i>Plasmodium yoelii</i> CU allele frequency of two independent genetic crosses grown in (a,b) naïve mice, (c,d) 17X1.1pp immunized mice and (e,f) CU-immunized mice. Light gray dots represent observed allele frequencies. Dark gray dots represent allele frequencies retained after filtering. Dark blue lines represent a smoothed approximation of the underlying allele frequency; a region of uncertainty in this frequency, of size three standard deviations, is shown in light blue. A conservative confidence interval describing the position of an allele evolving under selection is shown via a red bar. Allele frequencies are shown in log scale. <b>(B)</b> Evolutionary models fitted to allele frequency data. Filtered allele frequencies are shown as gray dots, while the model fit is shown as a red line. Dark blue and light blue vertical bars show combined and conservative confidence intervals for the location of the selected allele as reported in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006447#ppat.1006447.t003" target="_blank">Table 3</a>. Numbers in parentheses equate figures with locations in <b>(A)</b>. A black vertical line shows the position of a gene of interest.</p